The thyrotropin receptor (TSHR), expressed on the plasma membrane of thyroid epithelial cells as well as a variety of extra-thyroidal sites, is central to the regulation of thyroid growth and function. The TSHR is also the major autoantigen in the autoimmune hyperthyroidism known as Graves' disease where T cells and autoantibodies are directed at the TSHR. Our laboratory first demonstrated the existence of multimeric TSHR forms in native thyroid tissue and more recently we modeled such forms in transfected cells by using functionally tagged TSHRs and fluorescence resonance energy transfer (FRET). This competitive renewal application continues these studies with four specific aims: Specific AIM 1 - To determine the transmembrane contact sites of TSHR multimers. In previous studies we have determined that Y110 in the TSHR ectodomain is an important contact site for ectodomain dimerization and are currently pursuing complex mutational approaches, based on detailed molecular modeling, to determine the sites within the transmembrane domain which are responsible for the major multimeric forms of the full length TSHR. Specific AIM 2 - To determine the role of TSH ligand in TSH receptor multimerization.. Our preliminary data have suggested that TSH reduces TSHR multimers in FRET and Co-IP experiments but the mechanism for this effect is unclear. We will, therefore, further evaluate the influence of TSH induced regulation of the TSHR using bioinformatic and biochemical approaches. Specific AIM 3 - To illustrate that TSHRs reside within lipid rafts, are TSH ligand regulated, and are primary centers of signal initiation. Our data indicate that TSHR forms can be found in lipid raft compartments. Since lipid rafts are centers for signal transduction, we hypothesize that TSHRs within lipid rafts are major signal initiators and will examine this concept in detail. Specific AIM 4 - To test the hypothesis that TSHRs in lipid rafts are resistant to internalization. Internalization leads to the termination of signal transduction and would normally be expected to control excessive hyperstimulation as seen in Graves' disease. Internalization of TSHRs is regulated by the binding of -arrestin to phosphorylated receptors. Lipid rafts are centers of arrestin binding and in other receptor systems have been found to reduce the speed of receptor internalization. This may prolong receptor signaling which can have profound consequences in thyroid disease. We will therefore perform a detailed analysis including time-lapse imaging, to determine the role of arrestin and the signaling consequences. Significance Of the study: The overall aim of this grant is to understand the structure-function relationship of TSH receptor multimers and their role within and without lipid rafts. Understanding the biology of TSHRs may lead to better strategies to control excessive TSHR activation seen in human disease. Relevance: Thyroid disease is common amongst our VA patients and affects some 10% of the population with increasing prevalence with age. Autoimmune thyroid disease, while most common in women, often affects men to a worse degree. This is particularly true for Graves' eye disease. Hence, studies to investigate the cause of autoimmune thyroid disease are highly relevant to the mission of the VA and in addition serve as a model for all other autoimmune diseases including rheumatoid arthritis, multiple sclerosis and Type 1 Diabetes.